Mut^s型基因重组巴斯德毕赤酵母高密度发酵过程中甲醇流加的控制

Methanol Feeding Control in High-Density Cultivation of Mut^s Recombinant Pichia pastoris

在采用Muts 表型基因重组巴斯德毕赤酵母 (Pichiapastoris)发酵生产血管生长抑制因子(angiostatin)的过程中 ,诱导表达阶段甲醇的质量浓度对血管生长抑制因子的表达至关重要 .因重组Muts 型巴斯德毕赤酵母利用甲醇极慢 ,按文献方法流加甲醇时 ,甲醇逐渐积累达 30 g/L ,血管生长抑制素表达水平仅为 4mg/L .采用甲醇检测与控制系统对甲醇流加进行反馈控制后 ,甲醇质量浓度可稳定控制在设定范围 .采用此系统控制诱导阶段的甲醇流加 ,同时手动流加适量甘油以促进细胞生长 ,血管生长抑制因子表达水平量最高可达 89mg/L .

In the induction phase of fed batch cultivation of recombinant Pichia pastoris with a Mut s phenotype, methanol concentration is significant for angiostatin expression. Due to the extremely slow methanol utilization by this Mut s strain, methanol was accumulated to 30 g/L if it was continuously added based on the feeding strategies reported by other reference, and the level of angiostatin was only 4 mg/L. A methanol monitoring and control system was adopted for feedback control of methanol concentration , and the methanol concentration can be controlled at a predetermined level. During the expression phase, methanol concentration was controlled with this feeding system, at the same time glycerol was also added either continuously or manually in order to stimulate the cells' growth. In this way, the highest angiostatin concentration of 89 mg/L was reached.